This invention relates to a novel chondromodulin protein. More particularly, it relates to chondromodulin-I protein capable of stimulating the growth of chondrocytes in the presence or absence of fibroblast growth factor and promoting the differential potency of said cells, an isolated DNA (gene) encoding said protein, expression vectors containing said DNA, transformants capable of producing recombinant chondromodulin-I protein, a process for producing chondromodulin-I protein by culturing said transformants and a pharmaceutical composition containing chondromodulin-I protein as an active ingredient. The present invention also relates to the use of chondromodulin-I protein in the treatment of fracture and various cartilage diseases and as an anti-tumor drug.
Almost all the bones of mammals, except for flat bones such as cranial bone and the like, are formed through a mechanism called "intracartilaginous ossification", which comprises expression of primordial chondrocytes during the embryonic stage, growth and differentiation of said cartilaginous cells, generation of primordial cartilages such as proteoglycan, collagen II, collagen IX, collagen X and the like, infiltration of capillary vessels which is accompanied by the decomposition of ground substance of cartilage and progression of calcification around the vesicles of said ground substance, and the replacement thereof with bone as the final step. Thus, the cartilage metabolism plays a significantly important role in the bone-formation, especially in the elongation of a bone along the axis.
It has been known that a variety of hormones and growth factors participate in the bone-formation (osteogenesis) process, including insulin-like growth factor (IGF1, IGF2), fibroblast growth factor (FGF), growth hormone, tumor cell growth factor (TGF-.beta.) and the like. It has also been suggested that a certain active factor exists in cartilage, which stimulates the growth and differentiation of chondrocytes. However, there have been no reports which disclose the purification of the factor to such an extent that a single band is obtained on SDS-PAGE, and its definite physiological activity has not been determined. Neam et al. [Peter J. Neam et al., Journal of Biological Chemistry Vol.265, No.17, 9628-9633, (1990)] reported that they separated from bovine cartilage a sugar protein having an amino acid sequence highly similar to that of chondromodulin in the course of their studies for the identification of constitutive proteins in cartilage. However, they still have not elucidated the biological functions of said sugar protein.
The protein which concerns to the above-mentioned growth of chondrocytes and the like is known as chondromodulin protein having biological activities as illustrated below.
The expression of the growth and differentiation of chondrocytes plays an important role in the course of recovery from fracture or various cartilage diseases as follows: inflammatory reaction at the injured site, growth of the periost-derived cells, expression and growth of chondrocytes, synthesis of extra-cellular ground substances, calcification of said substances, and replacement thereof with bone tissues. As can be easily understood, the growth of cartilage tissue at the site of fracture is essential for the formation of bone tissue. Additionally, it is obvious that the growth of the chondrocytes is also important in the course of the recovery from cartilage diseases accompanied by cartilage destruction or injury. Furthermore, before the growth or metastasis of tumor cells, infiltration of blood vessels into tissues occurs for the supply of necessary energy to tumor cells, and therefor the inhibition of such a infiltration is thought to be effective for the prevention of growth or metastasis of tumor cells.
As can be seen from the above, it is desirable to obtain enough amount of chondromodulin protein capable of inhibiting the amplification of chondrocytes or infiltration of blood vessels and thereby providing treating methods effective on the above-mentioned diseases. However, industrial production of said protein from cartilage tissue was extremely difficult and practical application of said protein has been hindered because of the lacking of means to obtain the protein in large amount.
The present inventors have studied extensively with the aim of producing a large amount of chondromodulin protein by the use of recombinant DNA techniques and have now succeeded in the isolation of a novel protein, which belongs to a family of chondromodulin protein, useful for the establishment of the purpose of the invention, cloning of a gene (cDNA) encoding said protein, construction of an expression vector, transformation of heterogeneous cells, and production of recombinant protein by culturing the transformants. The present inventors also investigated physiological activities of thus obtained chondromodulin-I protein and demonstrated that said protein possesses above-mentioned activities and are useful in the treatment of fracture, various cartilage diseases, cancers and the like and can be formulated into pharmaceutical compositions.